Gasping for air: HIF2α in asthma

Abstract

Despite protective roles in various type of infection and in would healing, T helper (Th)2 cells are drivers of inflammation in allergic asthma. In this issue of Immunity, Zou et al. demonstrate the crucial involvement of hypoxia inducible factor (HIF)2α in promoting the differentiation of inflammatory Th2 cells, suggesting HIF2α as a promising therapeutic target for the treatment of allergic asthma.

Allergic asthma in both children and adults is a growing health concern worldwide, and understanding the mechanisms that promote inflammation in response to allergens is a critical step toward improved treatment and prevention. T helper (Th)2 cells form in response to a diversity of antigens including helminth parasites, common allergens (pollen, dust mites), and certain toxins like snake venom, leading to the production of type 2 cytokines that facilitate the body’s response against these threats.¹ This diversity suggests that phenotypic and functional heterogeneity in the Th2 population must exist in response to different stimuli.² In this issue, Zou et al. use single-cell datasets from both human and mice to identify Th2 cell populations driving allergic asthma.³ This analysis identifies a pathogenic population of Th2 cells with increased expression of the gene EPAS1 (Epas1 in mice) which encodes the protein hypoxia inducible factor 2a (HIF2α). Hypoxia is increased in the airways of asthma patients, suggesting that the low-oxygen environment of the lung may promote activation of HIF2α.⁴ Using mouse models of allergic asthma to test the role of HIF2α in Th2 cell generation, the authors uncover a HIF2α-GATA3 circuit which enhances the PI3K-AKT pathway through phospholipid metabolism to promote the differentiation of a pathogenic Th2 cell population that emerges from a TCF1⁺ stem-like population leading to increased airway inflammation and features of allergic asthma (Figure 1).

Figure 1. HIF2α promotes development of pathogenic Th2 cells in allergic asthma.

HIF2α activity promotes the differentiation of a Ly108⁺TCF1⁺ stem-like Th2 cell population to develop into CD25⁺ST2⁺ pathogenic Th2 cells through cooperation with GATA3 to induce inositol polyphosphate multikinase (IPMK) which enhances PI3K-AKT signaling via TCR. Blockade (PT-2385) or the absence of HIF2α induces cells to choose an alternate differentiation pathway that leads to less airway inflammation by forming Ikzf2⁺ Th2 cells and limiting formation of pathogenic Th2 cells.

Th2 cells orchestrate type 2 immune reactions through the secretion of interleukin (IL)-4, IL-5, and IL-13. Prolonged type 2 responses in the lung to allergens lead to asthma, necessitating treatment with steroid immunosuppressants. The transcription factor GATA3 is a central regulator of Th2 cells, and various molecules facilitate Th2 cell differentiation by co-operating with or supporting GATA3.⁵ While immunotherapeutics targeting type 2 cytokines show promise, a significant proportion of asthma patients exhibit poor responsiveness, indicating that the pathogenesis of asthma involves more than just cytokine production by Th2 cells. Zou et al. set out to examine the heterogeneity of Th2 cells by detailed analysis of single-cell RNA-sequencing data from both asthmatic mice and human subjects. These analyses revealed increased expression of EPAS1 (Epas1 in mice), the gene encoding HIF2α, in Th2 cells. HIF2α has known roles in the immune system, including regulation of macrophage polarization, neutrophil function, and the generation of regulatory T (Treg) cells, suggesting it may have a function in Th2 cells.⁴ In an OVA-induced murine asthma model, mice bearing T cells deficient in HIF2α (Epas1) exhibited less airway inflammation and reduced immune cell infiltration. Notably, loss of HIF2α reduced numbers of Th2 cells, as well as amounts of IL-4, IL-5, IL-13, and IgE in bronchoalveolar lavage fluid (BALF) and lung tissues. No significant alterations were observed in T_FH, Th17, and Treg cell populations between wild-type and HIF2α-deficient mice indicating a specific effect on Th2 cells. To better understand how HIF2α was contributing to Th2 cell differentiation, the authors performed single-cell RNA sequencing (scRNA-seq) on T cells from intact and HIF2α-deficient mice. Loss of HIF2α resulted in a decrease in the proportion of a specific cell cluster, characterized by high expression of genes Gata3, Il1lrl1 (which encodes the IL-33R ST2), Il5, Il13, and Pparg. In contrast, a population of cells expressing the transcription factor Ikzf2 increased in the absence of HIF2α. Furthermore, a stem-like population that expressed both the transcription factor TCF1 and Ly108 was more prominent in the absence of HIF2α. These HIF2α-dependent alterations in states of Th2 cells in the lungs and BALF indicated HIF2α’s role in promoting the process of differentiation toward a pathogenic state. Using pseudotime trajectory analysis, the authors observed that naive T cells undergo a transition from a stem-like stage toward one of two diverging pathways either into pathogenic (ST2⁺CD25⁺) or Ikzf2⁺ Th2 states. Furthermore, cell transfer studies confirmed the inability of HIF2α-deficient cells to become pathogenic Th2 cells, instead becoming the Ikzf2⁺ population of Th2 cells.

The transition from the stem-like Ly108⁺TCF1⁺ state to the pathogenic ST2⁺CD25⁺ state involved a simultaneous increase in HIF2α and GATA3 expression, indicating a coordinated role of these transcription factors. Indeed, the absence of HIF2α reduced GATA3, and genome analysis identified HIF2α binding sites on the Gata3 promoter, corroborated by luciferase assays and ChIP analysis, supporting a direct regulatory mechanism. Additionally, GATA3 modulates HIF2α expression, establishing a positive feedback circuit for these two transcription factors to promote Th2 cell differentiation. scRNA-seq and CUT&Tag analysis unveiled the collaborative regulation of key genes in the differentiation of pathogenic Th2 cells by HIF2α and GATA3. Pathway analysis further demonstrated their coordination in the modulation of phosphatidylinositol metabolites in Th2 cells, with HIF2α playing a pivotal role in the activation of the PI3K-AKT pathway during Th2 cell differentiation. By focusing on key genes co-regulated by HIF2α and GATA3 in the phospholipid pathway, the authors identified Ipmk, the gene encoding inositol polyphosphate multikinase (IPMK) which converts PIP₂ to PIP₃ activating the PI3K-AKT pathway. Indeed, Th2 cells deficient in HIF2α displayed reduced expression of IPMK, leading to impaired PI3K-AKT activation. Furthermore, overexpression of IPMK into HIF2α-deficient T cells restored Th2 cell differentiation. Thus, HIF2α and GATA3 coordinately support IPMK which increases AKT stimulation downstream of TCR stimulation leading to the pathogenic state of Th2 cells that promotes airway inflammation.

Collectively, these data identify a pivotal role of HIF2α in driving the pathogenic differentiation of Th2 cells in asthma, pinpointing it as a potential therapeutic target. Excitingly, administration of PT-2385, a specific inhibitor of HIF2α led to a substantial reduction in the frequency of Th2 cells and expression of Th2-associated genes in a murine allergic asthma model, aligning with observations in HIF2α-deficient mice. Moreover, PT-2385 treatment attenuated the presence of pathogenic Th2 cells and decreased pathway enrichment scores linked to type 2 immune responses, cytokine production, PI3K activation, and TCR signaling in both pathogenic and stem-like Th2 cells. Thus, HIF2α inhibitors such as PT-2385 exhibit potential as a therapeutic agent for asthma by targeting HIF2α-driven Th2 cell inflammation.

One area implied but left unaddressed was the timing of HIF2α’s role in the Th2 cell response and the necessity of hypoxic airways to promote HIF2α activity. Murine models in this study relied on systemic (i.p.) priming with OVA antigen prior to inhalation, implying that HIF2α impacts effector Th2 cell differentiation only in the lung, but perhaps not the initiation of the Th2 cell response when allergen sensitization occurs. Furthermore, it is unclear if HIF2α activation requires a hypoxic lung environment or whether it can perform these functions regardless of the oxygen status of the airways. Future research aimed to investigate the impact of HIF2α signaling in Th2 cells across various tissues and disease contexts beyond asthma would be of interest. This study supports previous research indicating that HIF2α deficiency does not affect Treg cell development.⁶ However, Treg cells lacking HIF2α demonstrate a reduced ability to suppress effector T cell-induced colitis and airway hypersensitivity, suggesting additional work may be important to resolve the heterogeneity of HIF2α-deficient Treg cells in conditions such as asthma. Furthermore, HIF2α signaling in hypoxic airways in lung epithelial club cells leads to ILC2 activation and inflammation.⁷ This suggests targeting HIF2α may have roles in several cells in hypoxic airways. Finally, the HIF2α inhibitor PT-2385 has shown promise in inducing tumor regression as a potential therapeutic option for treating renal cell carcinomas (RCC).⁸ Leveraging the findings from this study, there is a prospect of repurposing PT-2385 to alleviate asthma by modulating immune responses associated with airway hyper-responsiveness and inflammation. However, considering the chronic nature of asthma, a comprehensive evaluation of the long-term safety and efficacy profile of PT-2385 is crucial. Collectively, the findings by Zou and colleagues bring to light another important co-regulator of GATA3 and Th2 cell development by identifying HIF2α as a driver of Th2 cell differentiation in allergic asthma. In addition, their work highlights the heterogeneity of the Th2 cell response to allergens while providing a new therapeutic target in HIF2α that may provide an exciting opportunity for clinical treatment of patients with allergic asthma.